Major advances have recently been made in pharmaceutical technology to research new methods for the preservation of the intrinsic activity of polypeptides and to render them absorbent. Formulations able to ensure a reproducible absorption of these active molecules have the advantage of lacking side effects, unlike synthetic polymers. Of all the most widely used natural polymers the category of acidic polysaccharides is of particular interest. One of these, hyaluronic acid, a polysaccharide widely distributed throughout animal organisms, is constituted by units of D-glucuronic acid and N-acetyl D-glucosamine in alternate order. Its molecular weight can vary according to the methods used for its extraction and/or purification (EP 0138572 reg. on Jul. 25, 1990; EPA 0535200 published on Apr. 7, 1993; PCT Application Ser. No. WO 95/04132 published on Feb. 9, 1995; PCT Patent Application Ser. No. WO 95/24497 published on Sep. 9, 1995).
Besides the polymer's chemical-physical properties, the release methods and systems for biologically active molecules are also particularly important, such as microspheres which seem to be among the most versatile release systems. EPA 0517565 discloses a process for the preparation of microspheres, whose dimensions range between 1-100 μm, wherein the polysaccharide ester dissolved in an aprotic solvent such as DMSO, is added to a mixture of a high-viscosity mineral oil containing a non ionic surface active agent and ethyl acetate, which is a solvent for DMSO and the mineral oil, but not for the polysaccharide ester, which therefore precipitates in the form of microspheres having therefore the above mentioned dimensions.
Today, various techniques are known which involve the use of supercritical fluids for the production of finely subdivided particles with a narrow granulometric distribution curve. The supercritical antisolvent process is generally performed at moderate temperatures and enables the solvent to be completely removed from the precipitation environment. The applications concern substances that are heat-sensitive or difficult to handle, such as explosives (Gallagher. P. M. et al. 1989, Supercritical Fluid Science and Technology—Am. Chem. Soc. 334-354). Other applications concern the production of polymers in the form of fibers (Dixon, D. J. et al, 1993, J. Appl. Polym. Sci. 50, 1929-1942) and in the form of microparticles, including microspheres (Dixon, D. J., et al., 1993, AIChE J., 39, 1, pp 127-139). In the pharmaceutical field, the main interest is in the treatment of proteins (Tom, J. W., et al. 1994, Supercritical Fluid Engineering Science, pp 238-257, ACS Symp. Chap. 19, Ed. H. Kiran and J. F. Brennecke; Yeo, S. D., et al, 1993, Biotech, and Bioeng., 41, pp 341-346) and biodegradable polymers, such as poly(L-lactic acid) (Randolph, T. W., et al. 1993, Biotechnol. Prog., 9, 429-435; Yeo, S. D., et al, 1993, Macromolecules, 26, 6207-6210). Various methods have been devised for precipitation with a supercritical antisolvent. The semi-discontinuous method (Gallagher et al., 1989), involves injection of the antisolvent in the liquid solution which has already been prepared in the desired working conditions. The operation must be performed in a stepwise fashion to ensure that the liquid is removed, the final quantities of product are very limited and the spheres measure far more than 1μ in size.
Precipitation with a compressed antisolvent (PCA) involves injection of the solution in the high-density supercritical fluid (SCF) (Dixon et al., 1991; Dixon and Johnston, 1993). The injection times are much reduced to guarantee complete dissolution of the liquid, so the quantity of precipitate is very low, giving microfibers with an ordered structure.
The continuous process (Yeo et al., 1993a) enables the solution and the antisolvent to be injected simultaneously in the precipitation environment; the liquid expands and evaporates in the continuous phase, constituted by the SCF. The solution is injected through a micrometric nozzle with a diameter ranging between 10 and 30 μ. Solutions must be diluted to avoid blocking the nozzle and to prevent reticulate structures being formed. Consequently, the quantity of solid solute injected is very low. Moreover, a high ratio between the volume of antisolvent and solution must be used to continuously remove the liquid solvent from the precipitation vessel.
When the solution is placed in the precipitator and the container is loaded by means of SCF up to the desired pressure, the process assumes a completely discontinuous character (Yeo et al., 1993 a,b). By this technique, microspheres with a diameter of over 1 μ have been obtained. All the methods described here are accompanied by a final washing step to prevent the precipitate being resolubilized by the solvent. However, none of the cited techniques has been specifically applied to the production of high-molecular-weight biocompatible polysaccharide polymers and in particular the HYAFFs, namely the ester of hyaluronic acid, which are obtained by the procedure described in U.S. Pat. No. 4,851,521.